The chief goal is to understand how the hormone vasopressin regulates water excretion by the kidney. Vasopressin's action is mediated through regulation of the molecular water channel aquaporin-2. Based on our studies a decade ago, it is now clear that vasopressin regulates aquaporin-2 in a time frame of seconds to minutes by altering the distribution of the water channel aquaporin-2 between the plasma membrane and the cytoplasm via vesicular trafficking. Trafficking of aquaporin-2 to the plasma membrane renders the cells permeable to water. We are presently using a systems approach to address the mechanisms involved. For this approach, we are integrating protein mass spectrometry, DNA microarrays, mathematical modeling and physiological methods. The following is a summary of work over the past year appearing in the 14 references published from September, 2008 until July, 2009. The first 2 references in the reference list below show publications that have used protein mass spectrometry (1-2) to investigate protein networks involved in regulation of renal water and solute transport, as well as the mass spectrometry tools developed for these studies. The next 4 references (3-6) deal with studies of vasopressin action in regulation of aquaporin-2 using conventional physiological technques. The next 2 (7-8) are clinically-oriented papers which describe bioengineering work to exploit our recent discovery that normal kidneys excrete exosomes in the urine. Exosomes are are small membrane particles secreted by every cell type facing the urinary space in the kidney. The goal of these studies is to develop the methods infrastructure to allow clinical investigators to isolate urinary exosomes for disease biomarker studies. The next 3 references describe work focusing on the use of animal models of disease processes to discover the pathophysiological basis of salt and water imbalance disorders (9-11). The 12th paper describes a systems biology article investigating the transcriptional network involved in regulation of Aqp2 gene expression. The 13th and 14th articles are reviews. References 1. Zwang, N, Hoffert JD, Pisitkun T, Moeller HB, Fenton RA, Knepper MA. Identification of phosphorylation-dependent binding partners of aquaporin-2 using protein mass spectrometry. J Proteome Res. 2009 Mar;8(3):1540-54. 2. Sachs AN, Pisitkun T, Hoffert JD, Yu MJ, Knepper MA. LC-MS/MS analysis of differential centrifugation fractions from native inner medullary collecting duct of rat. Am J Physiol Renal Physiol. 2008 Dec;295(6):F1799-806. 3. Moeller HB, Knepper MA, Fenton RA. Serine 269 phosphorylated aquaporin-2 is targeted to the apical membrane of collecting duct principal cells. Kidney Int. 009 Feb;75(3):295-303. Epub 2008 Oct 8. PubMed PMID: 18843259. 4. Pisitkun T, Jacob V, Schleicher SM, Chou CL, Yu MJ, Knepper MA. Akt and ERK1/2 pathways are components of the vasopressin signaling network in rat native IMCD. Am J Physiol Renal Physiol. 2008 Oct;295(4):F1030-43 5. Moeller HB, MacAulay N, Knepper MA, Fenton RA. Role of multiple phosphorylation sites in the COOH-terminal tail of aquaporin-2 for water transport: evidence against channel gating. Am J Physiol Renal Physiol. 2009 Mar;296(3):F649-57. 6. Hoffert JD, Fenton RA, Moeller HB, Simons B, Tchapyjnikov D, McDill BW, Yu MJ, Pisitkun T, Chen F, Knepper MA. Vasopressin-stimulated increase in phosphorylation at Ser269 potentiates plasma membrane retention of aquaporin-2. J Biol Chem. 2008 Sep 5;283(36):24617-27. 7. Gonzales PA, Pisitkun T, Hoffert JD, Tchapyjnikov D, Star RA, Kleta R, Wang NS, Knepper MA. Large-scale proteomics and phosphoproteomics of urinary exosomes. Am Soc Nephrol. 2009 Feb;20(2):363-79. 8. Knepper MA. Common sense approaches to urinary biomarker study design. J Am Soc Nephrol. 2009 Jun;20(6):1175-8. 9. Li, JH, Chou CL, Li B, Gavrilova O, Eisner C, Schnermann J, Anderson SA, Deng CX, Knepper MA, Wess J. A selective EP4 PGE2 receptor agonist alleviates disease in a new mouse model of X-linked nephrogenic diabetes insipidus. J Clin Invest. 2009 Sep 1. Epub ahead of print PubMed PMID: 19729836. 10. Bockenhauer D, Feather S, Stanescu HC, Bandulik S, Zdebik AA, Reichold M, Tobin J, Lieberer E, Sterner C, Landoure G, Arora R, Sirimanna T, Thompson D, Cross JH, van't Hoff W, Al Masri O, Tullus K, Yeung S, Anikster Y, Klootwijk E,Hubank M, Dillon MJ, Heitzmann D, Arcos-Burgos M, Knepper MA, Dobbie A, Gahl WA, Warth R, Sheridan E, Kleta R. Epilepsy, ataxia, sensorineural deafness, tubulopathy, and KCNJ10 mutations. N Engl J Med. 2009 May 7;360(19):1960-70. 11. Jacob VA, Harbaugh CM, Dietz JR, Fenton RA, Kim SM, Castrop H, Schnermann J, Knepper MA, Chou CL, Anderson SA. Magnetic resonance imaging of urea transporter knockout mice shows renal pelvic abnormalities. Kidney Int. 2008 Nov;74(9):1202-8. 12. Yu MJ, Miller RL, Uawithya P, Rinschen MM, Khositseth S, Braucht DW, Chou CL, Pisitkun T, Nelson RD, Knepper MA. Systems-level analysis of cell-specific AQP2 gene expression in renal collecting duct. Proc Natl Acad Sci U S A. 2009 Feb 17;106(7):2441-6. 13. Hoffert JD, Chou CL, Knepper MA. Aquaporin-2 in the "-omics" era. J Biol Chem. 2009 May 29;284(22):14683-7. Epub 2009 Feb 4. Review. 14. Knepper MA. Physiology: Courier service for ammonia. Nature. 2008 Nov 20;456(7220):336-7.